Phosphorylation of eIF2α triggered by mTORC1 inhibition and PP6C activation is required for autophagy and is aberrant in PP6C-mutated melanoma

Abstract

Amino acid deprivation promotes the inhibition of the mammalian target of rapamycin (mTOR) kinase and activation of the general control nonrepressed-2 (GCN2) kinase. Signaling pathways downstream of both kinases have been thought to independently induce autophagy. Here we demonstrate that these two amino acid sensing systems are linked. We show that inhibition of mTORC1 leads to activation of GCN2 and phosphorylation of the eukaryotic initiation factor 2α (eIF2α) in a mechanism dependent on the PP6C phosphatase. mTORC1 inhibition does not lead to autophagy in the absence of PP6C activity, GCN2, or eIF2α phosphorylation. PP6C mutations commonly found in melanoma blunt the formation of a PP6C-GCN2 complex and are rapidly degraded, but paradoxically stabilize the wild-type PP6C allele and increase eIF2α phosphorylation. These PP6C mutations associate with increased autophagy in vitro and in vivo. Thus we have determined that phosphorylation of eIF2α by mTORC1 inhibition is necessary for autophagy and describe a novel role for PP6C in this system, which may be dysregulated in melanoma.

Synergistic inhibition of cell proliferation by combined targeting with kinase inhibitors and dietary xanthone is a promising strategy for melanoma treatment

“…The downstream kinases of PI3K/AKT signalling pathway are thought to independently induce autophagy . Previous studies showed that pharmacological inhibition of the PI3K–AKT–mTOR signalling pathway led to phosphorylation of the eIF2α, which is a marker of ER stress, and our results are consistent with those findings.…”

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

“…Although PP6 has an established role in TOR signaling in yeast [10], data confirming an analogous role in mammals has been scant. A recent report has linked PP6 to mTORC1 in the regulation of autophagy [13]. However, like our own results, these authors did not show that PP6 is directly responsible for the dephosphorylation of mTOR targets.…”

“…However, Wengrod et al [13] recently showed that PP6 is required for the pharmacological induction of autophagy by pharmacologic inhibition of mTORC1, an action that also required GCN2 (general control nonrepressed 2) and eukaryotic initiation factor 2α (eIF2α). As noted above, PP6 has been implicated in the regulation of cell growth and proliferation, which may reflect its involvement in signaling by the mammalian homolog of the Target of Rapamycin, mTOR.…”

Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

“…Biochemical characterization found that these mutations are heterogeneous with regards to their effect on PP6c activity, stability, and subunit binding. Consequently, the mutant enzymes can function as loss-of-function tumor suppressors or gain-of-function oncoproteins (17, 18, 28). In glioblastoma, the abundance of PP6c is increased and negatively correlated with patient survival (29).…”